Using single-molecule fluorescence in situ hybridization and immunohistochemistry to count RNA molecules in single cells in zebrafish embryos

Summary Taming gene expression variability is critical for robust pattern formation during embryonic development. Here, we describe an optimized protocol for single-molecule fluorescence in situ hybridization and immunohistochemistry in zebrafish embryos. We detail how to count segmentation clock RNAs and calculate their variability among neighboring cells. This approach is easily adaptable to count RNA numbers of any gene and calculate transcriptional variability among neighboring cells in diverse biological settings. For complete details on the use and execution of this protocol, please refer to Keskin et al. (2018),1 Zinani et al. (2021),2 and Zinani et al. (2022).3


SUMMARY
Taming gene expression variability is critical for robust pattern formation during embryonic development. Here, we describe an optimized protocol for singlemolecule fluorescence in situ hybridization and immunohistochemistry in zebrafish embryos. We detail how to count segmentation clock RNAs and calculate their variability among neighboring cells. This approach is easily adaptable to count RNA numbers of any gene and calculate transcriptional variability among neighboring cells in diverse biological settings. For complete details on the use and execution of this protocol, please refer to Keskin et al. (2018), 1

Institutional permissions
In this protocol, we used AB wild-type line, her1 ci301 ;her7 hu2526 and Df(Chr05:her1,her7,ndrg3a) b567 mutant lines. 2,4 The zebrafish experiments were performed under the ethical guideline of Cincinnati Children's Hospital Medical Center. The animal protocol was reviewed and approved by Cincinnati Children's Hospital Medical Center Animal Care and Use Committees (Protocol # 2020-0031). All zebrafish experiments must comply with protocols approved by a local animal ethics committee.
Order single-molecule fluorescence in situ hybridization (smFISH) probes To target specific mRNAs, order probes from ACDBio. Make sure they are assigned to different channels and compatible with other fluorophores that you are going to use for labeling. In this protocol, her1 and her7 mRNAs are labeled together with membrane-GFP by immunohistochemistry (IHC) and nucleus by Hoechst staining. Thus, ''Dr-her1-LE2-C3'' and ''Dr-her7-C1'' RNAscopeâ probes are ordered, respectively (Table 1).

KEY RESOURCES TABLE
Note: Adjust pH to 7.0 then filter in a sterile storage bottle by vacuum filtering with 0.22 mm pore. Note: Before start fixation, thaw one aliquot of 4% PFA and make sure it is at 20 C-25 C. Embryos would be around 10-11 somite stage at 9 am if they were incubated at 28 C until the shield stage and then incubated at 23 C for 12-16 h. Check GFP signal and morphology of embryos under fluorescence microscope. Remove embryos with abnormal morphology or weak GFP signal. Select 15 embryos for each experimental condition. Always keep embryos in dark; either cover tubes with aluminum foil or keep them in a dark box until the end of staining.

STEP-BY-STEP METHOD DETAILS
Note: Embryos would be ready to be fixed after removal of ones with abnormal morphology or weak GFP signal. But depending on the injection room temperature (21 C-28 C), embryos might be earlier than 10 somite stage at 9 am, and it might take 1-2 h to reach 12 somite stage.
2. Transfer embryos into 2 mL tube. Remove excess water. 3. Rinse with 1 mL 4% PFA (add and then remove 1 mL 4% PFA). 4. Add 2 mL 4% PFA. Cover the tube to keep embryos in dark and shake embryos for 1 h at 20 C-25 C. Note: Store the rest of 4% PFA at 4 C as it is going to be used in Day 3 and Day 6.
Note: Before the end of fixation (after $45 min), check transparency of embryos under dissecting microscope with dim light and from the black side of the mirror (see Figures 1A and 1B). Continue experiment only if the embryos are transparent (see Figure 1C). If the embryos are not transparent, it means the PFA is not good (see Figure 8B).
Note: In the meantime, mix 3 mL PBST-0.1% and 3 mL Methanol into a 60 mm petri dish.
7. Dechorionate embryos in the petri dish (3 mL PBST-0.1% + 3 mL Methanol) for 5 min in a dark room with the weakest microscope light. 8. Transfer embryos back to the tube. Rinse them with 1 mL Methanol. 9. Add 2 mL Methanol. Shake slowly for 5 min. 10. Refresh medium with 2 mL Methanol. Store tube for 24 h at À20 C.

Timing: 4 h
This section describes the steps for smFISH probe hybridization developed from the RNAscope technology and Gross-Thebing et al. 5 Note: Start this step afternoon ($1 pm). Set the oven to 40 C. Take tube out, wait until it reaches to 20 C-25 C.
11. Transfer embryos to 1.5 mL flat bottom microcentrifuge tube with glass pipette.
Note: Using 1.5 mL flat bottom microcentrifuge tube is important for evenly staining of 15 embryos.
Note: Using glass pipette is important as embryos might get stuck to the side of plastic tips.
12. Remove methanol as much as you can. Do not touch / damage embryos.

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Note: To remove solution as much as you can, first remove solution with 1000-mL filtered tip until less than 250 mL of the solution remains. Then hold the tube tilted, and remove the rest of the solution along the side of the tube taking care to avoid touching the embryos.  Figure 1D).
CRITICAL: Don't let the embryos dry. If embryos' color turns into yellow-orange color, which means they are dried, it becomes harder to flat mount embryos later.
14. Add 1 drop of RNAscope Protease-III pre-treatment solution. Gently tap the tube to sink embryos down. Wait for 20 min at 20 C-25 C.
CRITICAL: Protease makes embryos so fragile. After this step, be more careful and gentle with handling embryos till the end of experiment. From now on, never touch embryos with a tip while removing solutions. Add new solutions slowly from the inner side of the tube with $90 pipette tip (see Figure 1E). Always use either filtered or autoclaved tips. Shakings should be done at the slowest speed (i.e., approximately 20 rpm).

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Optional: You can prepare PBST-0.01% and fresh prehybridization buffer before starting the 15. Rinse with 1 mL PBST-0.01% gently. 16. Add 1 mL PBST-0.01%. Shake tube slowly in a horizontal position for 5 min (see Figure 1F). 17. Repeat the previous step for 2 more times. 18. Remove all solution, add fresh 250 mL prehybridization buffer. Place tube in a box with 45 tilted position (see Figure 1G) and incubate it in oven at 40 C for 2 h.
Note: Before 20 min end of prehybridization, take probes out from 4 C, warm them in oven (40 C) for 10 min, then cool to 20 C-25 C. Prepare probe mixture in a 1.5 mL microcentrifuge tube: 1 mL Dr-her1-LE2-C3 into 50 mL Dr-her7.
Note: While C1 probes are ready to use, C2 and C3 probes are shipped as a 503 concentrated stock. Thus, drop couple of droplets from C1 probe into a sterile microcentrifuge tube and use it as C1 aliquot.
CRITICAL: Before preparing the probe mixture, make sure all probes are completely dissolved.
19. Remove all solution. Add 15 mL probe mixture. Gently tap the tube. Place tube in a box vertically (see Figure 1H) and incubate it in oven at 40 C for 16 h. Note: Start probe hybridization at $6 pm and end it next day by $9 am to ensure sufficient hybridization and maintain consistency between each experiment.
Note: Add 1 mL probe mixture per embryo. If you have less than 10 embryo in a tube, add 10 mL probe mixture at a minimum volume. Do not include more than 15 embryos in a single tube.

Day 3: Amplifications and washes
Timing: 10 h This experimental day includes four signal amplification steps in between a series of washings. To detect target RNAs, probes are hybridized to amplification molecules, which make target RNAs to be visible in a fluorescent channel via binding of dye-labeled probes.
Note: Before starting this step, prepare 50 mL SSCT (0.23 SSC, 0.01% Tween 20). Place 4% PFA at 20 C-25 C. Be careful and gentle to embryos since they are fragile. Solutions should be added slowly from the inner side of the tube with $90 pipette tip (see Figure 1E). All the shakings should be done at the slowest speed ($20 rpm) at 20 C-25 C. Shake tube in a horizontal position (90 , see Figure 1F) if it contains $1 mL solution, in a tilted position (45 , see Figure 1G) if it contains 200-400 mL, or in a vertical position (0 , see Figure 1H) if it contains less than 200 mL.  Note: Take Amp 1 out to 20 C-25 C for 30 min before applying it.
28. Repeat the previous step for 2 more times. 29. Remove all SSCT.
CRITICAL: Remove all solution before amplification reagents to ensure staining quality and comparability.  Alternatives: Choose one of the alternate fluorescent color modules (Amp 4 Alt A, B, or C) that you want to label probes with different fluorophores (see Table 2). In this experiment, since we stain nucleus with Hoechst and membrane with GFP, we choose Amp 4 Alt B to label C1 (her7) and C3 (her1) probes with red and far red, respectively (see Table 1   Note: In order to avoid air bubbles, start preparing PBSTX one day before immunohistochemistry staining. Mix 5 mL 103 PBS with $30 mL DEPC water, then add 500 mL Triton X-100. Mix well and leave it at 20 C-25 C. Top it up to 50 mL with DEPC water on the next day.

Day 4: Primary antibody staining
Timing: 4 h The following steps describe primary antibody staining for IHC on the fourth day of experiment.
Note: Start this step afternoon ($1-2 pm). Top up PBSTX to 50 mL that was prepared the day before. Final concentration would be 13 PBS 1% Triton X-100.
CRITICAL: Do not start too early or late to make sure enough incubation time and to finish the last step at around 6 pm as embryos are very fragile and buffers are pretty harsh.
54. Repeat the previous step for 2 more times.   Note: Take 4% PFA and SSCT out and warm up to 20 C-25 C. 62. Remove secondary antibody solution. Rinse and wash embryos once with SSCT. Shake tube slowly in a horizontal position for 7 min at 4 C in a cold room. 63. Rinse and add 1 mL 4% PFA.
Pause point: Embryos can be stored in dark at 4 C for up to 7 days. 64. Place tube on ice. Use SSCT for sample preparation in a 60-mm petri dish.
CRITICAL: Avoid light as much as possible during sample preparation. Work in a dark room with the weakest microscope light. Turn microscope mirror to $90 degree against desk for a minimum light exposure.
65. Place one embryo into petri dish using a transfer pipette. First, cut from the head with a microsurgical knife. Then do little cuts on the skin to remove yolk from the tissue (see Figure 2A). Use a lash tool to gently remove excess yolk. Cut until the presomitic mesoderm (PSM) and 1-2 somites are left.
CRITICAL: Remove all yolk to ensure integrity of the PSM because yolk proteins interfere with fluorescent proteins.
66. Place one drop of ProLong Gold antifade reagent on a glass slide and transfer tissue into it using a lash tool (see Figure 2B). 67. Make sure the tissue is dorsoventrally flattened (see Figure 2C). 68. Apply transparent nail polish and then fix with the coverslip on top gently (see Figures 2D and  2E). Wait for 5 min for nail polish to dry before starting the imaging.
CRITICAL: Apply consistent amount of nail polish to control the PSM tissue thickness and imaging quality.
CRITICAL: Keep prepared slides in dark at 4 C, and complete imaging on the day of sample preparation.

Imaging
Timing: 12 h to 7 days This section describes steps and parameters for confocal imaging of flat-mounted smFISH and IHC stained samples (see Tables 3 and 4 for microscopy and channel settings).
Note: Resonant scanner confocal microscope is recommended to perform smFISH imaging. In our experiment, we used Nikon A1R HD confocal on TiE microscope with an Apo TIRF 1003 Oil DIC N2 objective. For an equal comparison, do not change laser settings in the channels for RNA probes among the samples. On the other hand, you can set different laser powers and voltage in membrane and nucleus channels in order to achieve better cell segmentation. For an even imaging quality, periodically check laser strength.
Note: Find appropriate laser settings for nucleus and membrane channels of each sample at the most anterior part of the tissue as the region of interest (i.e., PSM tissue) might be affected due to photobleaching. Signals should not be oversaturated but high enough to be detected in Imaris cell segmentation. In our case, 50-60 laser power and $60 PMT HV in DAPI channel, and 2-10 laser power and 10-20 in GFP channel depends on membrane injection and staining quality.

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CRITICAL: As GFP can bleach quickly, avoid high laser power.
Note: Since Channel 4 has a different integrate level, save it as a separate optical configuration in Nikon's confocal NIS-Elements software.
69. Put oil onto the lens of 1003 objective. 70. Place specimen onto the stage. 71. Find focus plane by ocular lenses. 72. Check nucleus (Hoechst) and membrane (GFP) signal at the most anterior part of the tissue and find appropriate laser power and PMT HV (see Figures 3A and 3B). 73. Open Acquire>Scan Large Image. Select ''Left, top, right and bottom limits'' in the Area panel. 74. Set stitching overlap as 5% (see Figure 3C). 75. In the DAPI channel (for nucleus), run the Live camera, locate sample's XY limits. Then, in the same channel, set the Z range at the positions where there is no signal by using Top and Bottom buttons in the Z panel in the ND Acquisition window. Thus all the tissue is covered in the image dorsoventrally. Press Home (middle Z position), go to symmetric mode and press Relative button (see Figure 3D). Then go to middle XY position and close ''Scan Large Image'' window.
Note: Keep in mind the number of X and Y fields to use it while adding custom XY multipoints in the following step.
76. Go to XY panel in the ND Acquisition window. Select ''include Z''. Press Custom button. Under the Large Image section, select 1003 objective, write number of X and Y fields, and 5% as overlap area (see Figures 3E and 3F).
Note: This step adds custom XY multipoints, which cover the tissue, at the middle Z layer.
Note: Deselect any multipoint if it doesn't include the tissue because of rotated sample mounting.
77. Add optical configurations in to the l (multichannel) panel in the ND Acquisition window (see Figure 3G).
Note: While one of the optical configurations includes DAPI-TRITC-Cy5 channels, the other one includes FITC channel because they have different signal integration parameters.

Analysis
Timing: 2 to 7 days This section describes steps to analyze images. First, prepare images for analysis (i.e., creating surface, masking and cell segmentation). Then, using cell positions and single transcript levels per cell, run ''variability (noise)'' codes for the RNA and variability distribution.
Masking and cell segmentation 79. Open multipoint ND2 file in NIS Elements. Go to ''Image > ND Processing > Stitch Multipoint to Large Image''. Stitch mutipoints via blending without shading correction. Save the stitched file (see Figure 4A). 80. Use ''Imaris File Converter'' to convert stitched ND2 file to IMS file (see Figure 4B). 81. Open IMS file in Imaris software. Use ''Surface'' wizard to mask nucleus and membrane of the tissue.  Figure 4C).
Note: If the tissue borders change dramatically along the z-axis (e.g., dorsal side of the tissue, where tissue narrows immediately), select the tissue borders in every 2-3 slices instead of 5.
CRITICAL: Select precisely from the border of the PSM cells. Make sure to include adaxial cells in the PSM and S1 somite, but exclude notochord, neural tube, and skin cells.
Alternatives: Select out of PSM and mask it out later. Note: Deselect ''Duplicate channel before applying mask''.
Alternatives: If you selected outside of the tissue, set voxel intensity inside surface to zero.
Note: Cell creation parameters can be stored and used for a batch analysis in Arena later.
CRITICAL: Embryos within the same batch should have similar quality score.

97.
After the end of cell segmentation (see Figure 5B), export all cell statistics to file in XLS format (see Figure 5C) from every split images (see Table 6 for required cell statistics).

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98. Add a ''Measurement Point'' in the Surpass Tree list of the left-anterior and right-anterior files. Find anterior borders of the PSM tissue in each sample (see Figure 5D). Create ''Somite'' sheet in all anterior files (i.e., ''TITLE_LA.xls'' and ''TITLE_RA.xls''). Add X-Y positions of two points in Cell A2-B2 for ''Point A'' and Cell A3-B3 for ''Point B'' in the ''Somite'' sheet (see Figures 5E and 5F). 99. Measure angle and distance from the tail end of expression stripes of the segmentation clock in the PSM in all embryos in each genetic background (see Figures 6A-6C). Then fit an equation to the data, i.e., ''Angle_vs_Distance = delta_angle * Distance + angle'', which provides ''delta_angle'' and ''angle'' to be used in ''GENOTYPE_wVol.py'' or ''GENOTYPE_woVol.py'' later for analysis (see Figure 6D).
CRITICAL: As the angles of stripes vary smoothly along the posterior-anterior axis, it is important to use comparable angles of slices with that of expression stripes.

LIMITATIONS
The smFISH technique described in this protocol is limited to simultaneously detect up to two different RNA targets per cell. Two additional channels are spared to detect nucleus and membrane for proper cell segmentation in a compact tissue. Injection of large quantities of membrane-localized fluorescent protein RNA might cause abnormal morphology in few embryos. So one should not inject too much RNA.

Potential solution
Fixing embryos properly with PFA is a critical step prior to smFISH staining. If it has a pH different than 7.0, or if it is an old solution, it causes non-transparent tissue, which leads to high background staining signal. Prepare fresh PFA with a pH 7.0. Aliquot into 15 mL tubes and store at À20 C. Always avoid multiple freeze and thaw cycles.

Problem 2
Yellow-orange colored embryos after air dry (Refer to day 2: probe hybridization, step 13).

Potential solution
Do not let embryos dry. It is enough to wait until there is still some methanol left at the very bottom of the tube, but embryos should lose their contacts through methanol.

Potential solution
After incubation of embryos with RNAscope Protease-III pre-treatment solution, embryos become more fragile. Be gentle after this step until the end of staining (day 6). Add solutions slowly (see Figure 1E). Use the lowest speed of rotary shaker. Do not touch embryos with pipette tip etc.

Problem 4
No signal in RNA channels after smFISH staining (Refer to imaging, step 71).

Potential solution
Always use filtered tips and RNase free solutions. Otherwise smFISH probes or RNAs might be degraded. Check probe hybridization and amplification steps (Refer to day 2 and day 3). Make sure embryos were at the bottom of the tube and covered by the solutions in each step. Tap tube gently to sink embryos if they are floating after adding amplification solutions.

Problem 5
Localized blockage of signal in the tissue (Refer to imaging, step 72).

Potential solution
If you image dorsoventrally, the yolk proteins might interfere with fluorescent proteins. Make sure you cleaned yolk from the tissue. Use soft lash tool. Do not damage the tissue while deyolking.

Problem 6
No or low GFP signal (Refer to imaging, step 72).

Potential solution
Make sure you injected enough amount of membrane-localized GFP RNA (400 pg) (Refer to before you begin, step 7). Pick embryos with a high GFP signal before fixation. Do not continue if you don't have GFP positive embryos (Refer to day 1: fixation, step 1). Do not excite the tissue too much to find appropriate laser power for FITC channel as GFP can bleach quickly (Refer to imaging, step 72).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to the lead and corresponding author Ertu grul M. Ö zbudak (Ertugrul.Ozbudak@cchmc.org).

Materials availability
This study did not generate new unique reagents.